The significance of glycosylation on the function of the calcium-sensing receptor (CaSR) dataset

Brief description

This dataset is an outcome of a series of experiments conducted to investigate the significance of glycosylation on the function of the human calcium-sensing receptor (CaSR). The CaSR is a family C G protein-coupled receptor that plays an important role in regulating whole body extracellular calcium (Ca2+o) homeostasis.  The exact location of the binding sites for Ca2+o (and other cations) is still unknown, but experimental and in silico analysis has provided evidence that they may be in the extracellular domain and the transmembrane domain. Post translational modifications leads to the covalent attachment of a number of negatively charged sugars, which may provide a negatively charged surface for polyvalent cations to interact with.

There are 11 potential N-linked glycosylation sites in the CaSR’s extracellular domain.  A single conservative asparagine to glutatmine mutation was undertaken to disrupt individual N-linked glycosylation consensus sequences.  Alanine scanning mutations were also used to assess the significance of potential O-linked glycosylation sites (small clusters of Ser/The residues near the membrane anchor points of the exo-loops in the transmembrane domain).  These mutant receptors were then transiently expressed in HEK293 cells. The impact of each mutation on cell expression and function was assessed by the following techniques:

• Western blotting: Hand cast SDS-PAGE gel and Membrane transfer was undertaken using the BioRad mini-protean system, Precast gradient gels (4 – 15%) were TGX precast gels from Biorad. Western blotting films were scanned in .JPG and .PNG format. Quantification of bands was undertaken using GNU Imaging Manipulation Program (GIMP) imaging software.

• Cell surface expression ELISAs: Antibody based enzyme-linked immunosorbent assays (ELISAs) were used. The chromogenic substrate for horseradish peroxidase (HRP) was 3,3’,5,5’-Tetramethylbenzidine (TMB). Absorbance at 450 nM was read using an EnVision Multilabel Reader and exported using .XLS files. Data were represented and analysed using GraphPad Prism 5.

• Microfluorimetry with fura-2 AM (a calcium-sensitive fluorophore): Fluorescent images were taken on a Zeiss Axiovert 200M microscope with a Carl Zeiss monochrome HSm digital camera using a 63x Zeiss long-distance objective. Excitation filters, emission filters and dichroic mirrors were all obtained from Zeiss. The fluorescent light source was provided by a Lambda Sutter DG-4 wavelength switcher. The ratiometric (340/380 nm) region-of-interest data were exported using Stallion software (.SLD files) and subsequently processed using GraphPad Prism 5.0.

The creation of the mutant cells and expression/function experiments were carried out by Dr Sarah Brennan and Karolina Windloch. Data analysis was undertaken by Dr Sarah Brennan. For further information, refer to the associated thesis.